This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. How does the brain work? In order to dilucidate function, structure must be inspected first. Our lab has concentrated efforts in the study of the Drosophila brain. The fruit fly offers, as a model system, ready-made tools for the specific labeling, hyperactivation and disruption of tissues and cells. With the help of these tools, our lab has generated high-resolution maps of the different elements of the larval fly brain throughout embryonic and larval stages of development. A combination of limitations in both confocal imaging and in the individual labeling of tightly positioned, minute structures such as dendritic spines and terminal neurite branches has hindered the inspection of the fine connectivity ("microcircuitry") of the brain. To achieve the required resolution for the elucidation of microcircuitry, we have started studies at the electron microscopic level. The first instar larval brain provides us with a small, yet functional (and developing) brain. Neuronal bodies are arranged along the cortex in lineages which project centripetally in bundles, which branch at constant positions and generate a fiber-only neuropil that extends into the ventral nerve cord. At about 1000 neurons and a neuropil volume of roughly 25x25x25 microns, the fine description of the first instar brain is a considerable, but doable, task given current technologies. We have performed ultrathin serial sections (60 nm) and imaged the neuropil of 330 sections at 11,000 magnification, covering about 23 microns. In addition, we have developed the custom software package TrakEM2 for the storage, visualization, annotation, measurement and 3D modeling of our data.